Phosphorylated paxillin and phosphorylated FAK constitute subregions within focal adhesions

Bachmann, Michael; Skripka, Artiom; Weißenbruch, Kai; Wehrle-Haller, Bernhard; Bastmeyer, Martin

doi:10.1242/jcs.258764

Cited by 8 publications

(9 citation statements)

References 88 publications

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“…S3). These also revealed the presence of clustered regions within focal adhesions similar to previous studies [46, 49–52].…”

Section: Resultssupporting

confidence: 90%

“…Here, we investigate this hypothesis and define key properties of nanoclusters which allows it to function as a signalling hub by understanding nanoclusters of transmembrane receptors, integrin. Focal adhesions comprise of nanoclusters of integrin and other adhesome proteins [1,30,46,[49][50][51][52]. However, are these nanoclusters constantly cycling or are they stable over time is not well understood.…”

Section: Discussionmentioning

confidence: 99%

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Nano-clusters of ligand-activated integrins organize immobile, signalling active, nano-clusters of phosphorylated FAK required for mechanosignaling in focal adhesions

Jain,

Minhaj,

Kanchanawong

et al. 2024

Preprint

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Transmembrane signalling receptors, such as integrins, organise as nanoclusters that are thought to provide several advantages including, increasing avidity, sensitivity (increasing the signal-to-noise ratio) and robustness (signalling above a threshold rather than activation by a single receptor) of the signal compared to signalling by single receptors. Compared to large micron-sized clusters, nanoclusters offer the advantage of rapid turnover for the disassembly of the signal. However, if nanoclusters function as signalling hubs remains poorly understood. Here, we employ fluorescence nanoscopy combined with photoactivation and photobleaching at sub-diffraction limited resolution of ∼100nm length scale within a focal adhesion to examine the dynamics of diverse focal adhesion proteins. We show that (i) subregions of focal adhesions are enriched in immobile population of integrin β3 organised as nanoclusters, which (ii) in turn serve to organise nanoclusters of associated key adhesome proteins-vinculin, focal adhesion kinase (FAK) and paxillin, demonstrating that signalling proceeds by formation of nanoclusters rather than through individual proteins. (iii) Distinct focal adhesion protein nanoclusters exhibit distinct dynamics dependent on function. (iv) long-lived nanoclusters function as signalling hubs-wherein phosphorylated FAK and paxillin formed stable nanoclusters in close proximity to immobile integrin nanoclusters which are disassembled in response to inactivation signal by phosphatase PTPN12 (v) signalling takes place in response to an external signal such as force or geometric arrangement of the nanoclusters and when the signal is removed, these nanoclusters disassemble. Taken together, these results demonstrate that signalling downstream of transmembrane receptors is organised as hubs of signalling proteins (FAK, paxillin, vinculin) seeded by nanoclusters of the transmembrane receptor (integrin).

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“…S3). These also revealed the presence of clustered regions within focal adhesions similar to previous studies [46, 49–52].…”

Section: Resultssupporting

confidence: 90%

Section: Discussionmentioning

confidence: 99%

Nano-clusters of ligand-activated integrins organize immobile, signalling active, nano-clusters of phosphorylated FAK required for mechanosignaling in focal adhesions

Jain,

Minhaj,

Kanchanawong

et al. 2024

Preprint

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show abstract

“…Paxillin has been shown to be recruited to integrins via kindlin (Theodosiou et al ., 2016) and talin (Pinon et al ., 2014), and talin-dependent paxillin recruitment is necessary to stabilize integrin adhesions (Ripamonti et al ., 2021). At the same time, paxillin interacts closely with FAK and their interaction regulates integrin dynamics (Bachmann et al ., 2022; Zaidel-Bar et al ., 2007; Ballestrem et al ., 2006). Vinculin supports force transmission between the integrin-talin-actin axis (Elosegui-Artola et al ., 2016; Humphries et al ., 2007; Thievessen et al ., 2013) and could be expected to facilitate αVβ6-mediated mechanical TGFβ1 activation.…”

Section: Discussionmentioning

confidence: 99%

New tools to study the interaction between integrins and latent TGFβ1

Bachmann

Kessler

Burri

et al. 2023

Preprint

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Transforming growth factor beta (TGFbeta) 1 regulates cell differentiation and proliferation in different physiological settings, but is also involved in fibrotic progression and protects tumors from the immune system. Integrin alphaVbeta6 has been shown to activate latent TGFbeta1 by applying mechanical forces onto the latency-associated peptide (LAP). While the extracellular binding between alpha;beta6 and LAP1 is well characterized, less is known about the cytoplasmic adaptations that enable alphaVbeta6 to apply such forces. Here, we generated new tools to facilitate the analysis of this interaction. We combined the integrin-binding part of LAP1 with a GFP and the Fc chain of human IgG. This chimeric protein, sLAP1, revealed a mechanical rearrangement of immobilized sLAP1 by alphaVbeta6 integrin. This unique interaction was not observed between sLAP1 and other integrins. We also analyzed alphaVbeta6 integrin binding to LAP2 and LAP3 by creating respective sLAPs. Compared to sLAP1, integrin alphaVbeta6 showed less binding to sLAP3 and no rearrangement. These observations indicate differences in the binding of alphaVbeta6 to LAP1 and LAP3 that have not been appreciated so far. Finally, alphaVbeta-sLAP1 interaction was maintained even at strongly reduced cellular contractility, highlighting the special mechanical connection between alphaVbeta6 integrin and latent TGFbeta1.

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“…Paxillin is a scaffold/adapter protein contributing to cell migration by regulating focal adhesion dynamics 38,53,54 . Previous work reported that paxillin phosphorylation is essential for the assembly of focal adhesion and lamellipodia protrusions formation 55,56 . Our study used a mAb against phospho-paxillin (Tyr31) for immunolabelling transfected cells with the dsFBXO32 condition.…”

Section: Discussionmentioning

confidence: 99%

Heterospecific developmental models highlight the role ofFBXO32in neural crest lineages and the significance of its variant in pediatric melanoma

Vargas

Guillermain

Gorojankina

et al. 2023

Preprint

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Melanoma is a rare sporadic pediatric cancer caused by de novo genetic accidents. To analyze the involvement of FBXO32 of unknown biological significance, we set up original developmental in vivo, ex vivo, and in vitro models to unmask the biological role of FBXO32 in cephalic and trunk neural crest lineages. We show that FBXO32 silencing impairs melanocyte proliferation, differentiation, and vascularization. In rescue experiments, co-electroporation with the human wild-type gene compensates for the silencing of the endogenous gene and restores normal melanocyte phenotypes. By contrast, co-electroporation with the human variant, FBXO32G139S, alters melanocyte differentiation, pigment synthesis, triggers migration to the dermis, and favors angiotropism. Transcriptomic analysis reveals targets associated with melanocyte transformation, cytoskeleton remodeling, and focal adhesion. We show that FBXO32 orchestrates unexpected interactions with ASIP for melanogenesis and BAP1 for the post-translational ubiquitination system. We propose FBXO32 as a tumor suppressor gene capable of preventing the onset of pediatric melanoma.

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Phosphorylated paxillin and phosphorylated FAK constitute subregions within focal adhesions

Cited by 8 publications

References 88 publications

Nano-clusters of ligand-activated integrins organize immobile, signalling active, nano-clusters of phosphorylated FAK required for mechanosignaling in focal adhesions

Nano-clusters of ligand-activated integrins organize immobile, signalling active, nano-clusters of phosphorylated FAK required for mechanosignaling in focal adhesions

New tools to study the interaction between integrins and latent TGFβ1

Heterospecific developmental models highlight the role ofFBXO32in neural crest lineages and the significance of its variant in pediatric melanoma

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